Fabrication and characterization of 3D printing scaffold technology by extract oils from plant and its applications in the cardiovascular blood

Extract oils from plants used in 3D polysaccharides modified with natural protein polymer modified polymer scaffolds can help to reduce blood pressure. This study aimed to use extract oils from plant (EOP)as blood pressure-reducing, bind them to magnetic iron nanoparticles (Fe3O4@NPs), then bind them to polymeric 3D print scaffolds [chitosan, polylactic acid, and polyurethane (CS/PLA/PU), modified with natural protein and finally separate them. This method made it possible to investigate different variables for nanoparticles. In this project, synthesis polymer, modified gelatin (Mo-Ge), PEGylation, extract oils from plant loading and release process in nanocarrier with different concentrations were examined and cell proliferation was optimized. The results show that 75% of the extract oils from plant loaded on iron magnetic nanoparticles containing PEGylated polymer scaffolds was released. Cell proliferation was performed for the sample. In this process, modification of scaffolding with polysaccharides modified with natural protein and extract oils from plant increased the efficiency of nanoparticles among the studied Allium sativum and Zingiber officinale. The size of A. sativum and Z. officinale were 29.833 nm and 150.02 nm size, respectively. These behaved very similarly to each other and A. sativum had the biggest effect in lowering blood pressure. The application of extract oils from plant in 3D mode scaffolding has not been studied before and this is the first analysis to do so, using nanoparticles.

properties of nanofibers in anticoagulant drugs. O'Brien 9 explained different materials application in scaffolds concerning the tissue engineering field. Tissue engineering with 3D printing can produce and repair damaged tissues engineering by combining cellular parts of the body with biocompatible materials. An et al., 10 explained that-compared to synthetic polymers 3D technical natural polymers can provide good biocompatibility for cells. The ability of natural polymers to print in 3D scaffolds is usually weak so for this reason, indirect 3D printing was created for porous 3D scaffolds. The reason of compositions of scaffold for the process, usage the abilities of each in this scaffolding. Two EOP of A. sativum and Z. officinale have been selected from among the eight EOP that are effective in regulating blood pressure 11 . A. sativum reduces the risk of heart disease by managing high cholesterol and blood pressure. Z. officinale can help lower blood cholesterol levels and prevent blood clots. By lowering cholesterol levels and blood clotting, the risk of clogged arteries is reduced and the risk of heart attacks and blood pressure. In fact, the main purpose of preparing blood vessels with suitable polymers in the form of 3D printing and placing drugs that regulate blood pressure on them. Among the EOP that are effective in regulating blood pressure, experiments were performed on eight EOP 12 . The results indicate that A. sativum and Z. officinale show the most similar behavior in regulating blood pressure, which confirms the similarity of the FT-IR and XRD spectra of these two drugs. Previous studies 13,14 have shown that they are similar in terms of therapeutic effects they can have on the body. Because the investigation of analysis spectra was more pronounced in the samples containing A. sativum and Z. officinale. The study continued our research on it. The amount of drug loaded and the amount of drug released were measured in the same way as in the previous article and good results were obtained 15 .
Synthesis of magnetic iron oxide nanoparticles. 307.2 g of hexahydrate of iron chloride, 97.7 g of short-grained iron chloride were dissolved in 100 ml distilled water to make magnetic NPs. The subsequent product was placed in a bathroom sonicator for 30 min, then stirred for 2 h under a stream of nitrogen gas. Concentrated ammonia was used as the precipitating agent. Following the reaction, the sediments were separated by a magnetic separation method with a 1.3 T magnet (and washed with distilled water and ethanol). The sediment was then dried in an oven at 40 °C.
Connection of A. sativum and Z. officinale to magnetic nanoparticles. In order to take advantage of the antihypertensive properties of EOP, various EOP were used, including olive oil, sesame, coconut, almond, A. sativum and Z. officinale, lavender and coriander. In the section used eight oil, but two oil suitable selected for the work. In this section, 0.5 g of eight each oil mixed with 0.5 g of Fe 3   XRD analysis. XRD (Siemens D 5000 X-ray Powder Diffraction System, Germany), is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions. The XRD analyzed material is finely ground, homogenized, and average bulk composition was determined. XRD using copper beams at 25 °C, at a rate of degrees per second, on a scale of 2θ and in one step of 1 s. The average crystalline size was obtained from X-ray diffraction data using Scherrer's formula (Eq. 1): where k = 0.94, λ = 0.154056 nm, and β is the full width at half maximum in radians 16,17 . FT-IR analysis. FT-IR microscope (Thermo Scientific Inc., USA) with liquid nitrogen cooled MCT-A detector has been used, conical shape germanium tip crystal (350-micron spherical finish, single reflection, throughput > 50%, 27° average angle). Each spectrum has been achieved in the range of 4000-650 cm −1 at a spectral resolution of 8 cm −1 and with 128 scans on the average using Omnic software (Thermo Sci-entific Inc., USA). Determination of EOP release profile. 40 mg of the EOP (A. sativum and Z. officinale) was dissolved in 100 ml of phosphate buffer solution with pH = 7.4 and stirred at room temperature with a magnetic stirrer. With a certain time, interval of up to 48 h, 5 ml from the top solution was removed and the sediments in it were separated. Then its absorption was read by a UV-Vis device at 275 nm and the amount of EOP was obtained from the calibration curve. The EOP cumulative percentage was calculated from the following equation (Eq. 3):  (Fig. 2C), identified the chemical absorption (Fig. 2). FT-IR spectrum of iron magnetic NPs connected to Z. officinale (Fig. 2D), after connecting with polymeric scaffolds (CS/PLA/PU) (Fig. 2E), and after the PEGylation (Fe 3 O 4 @Z. officinale/CS/PLA/PU-Mo-Ge) (Fig. 2F), identified the chemical absorption (Fig. 2). The results show that the A. sativum and Z. officinale exhibited the closest behavior to other oils (including olive oil, sesame, coconut, almond, A. sativum and Z. officinale, lavender and coriander). In Fig. 2A,D, the absorption of Fe-O of iron magnetic NPs is seen at 500 cm −1 . The specific peaks at 476 and 578 cm −1 may be due to O-Fe stretching vibration and 1645 cm −1 is related to H 2 O deformation, respectively 18  XRD analysis. For evaluate the appropriate particle size and morphology, XRD was used to confirm the results of SEM images. Figure 3A  www.nature.com/scientificreports/ shows the most obvious changes (Fig. 3A,B). In Fig. 3A, which refers to the XRD analysis of the sample containing A. sativum, it clearly shows the changes in increasing both the NPs and CS/PLA/PU, and PEGylating the sample (Fig. 3A). The XRD patterns exhibited peaks corresponding to  Figure 4B shows the SEM image of Fe 3 O 4 @A. sativum/CS/ PLA/PU, and it can be seen clearly that the particles are uniformly aggregated, spherical shaped with a size of 50-350 μm 2,29,30 . Figure 4B shows the PEGylation of the 3D print scaffold (CS/PLA/PU) attached to the magnetic iron NPs containing the gelatin-coated A. sativum. Figure 4C corresponds to the combination [(Fe 3 O 4 @A. sativum/CS/PLA/PU-Mo-Ge) PEGylated]. In the SEM image of iron magnetic NPs connected to Fe 3 O 4 @A. sativum, it can be seen clearly that the particles are uniformly aggregated, spherical shaped with a size of 6-30 nm 28 . In the section entered SEM of A. sativum. Because the SEMs of the A. sativum and Z. officinale oils were similar. In this study we attempted to examine the image with an electronic microscope. Since the sample was very thick and oily and did not dry completely even under vacuum, we had to dissolve it in a little methanol and then take a picture of it. Measurement of the sample when using SEM revealed that the sample was 100-135 nm in size.

ZPS analysis.
The ZPS represents the electric charge on the membrane surface. The ions in the environment as well as the pH of the environment affect ZPS amount. In this experiment, ZPS, deviation and guidance were obtained as − 1.29, 0, and 13.3, respectively. These values indicate that the polymers used in the construction of the scaffold have good conductivity in solution and are also blood compatible. Therefore, they can be effective in in vivo testing. The particle has a surface charge inside the fluid, and an increase in the concentration of ions with the opposite charge to the surface of the particle is always seen around the surface of the particle inside the fluid. Thus, an additional layer of these ions surrounds the surface of the particle and forms an additional layer around the particle. When a particle moves in a fluid, the surrounding layer also moves with the particle and moves with the particle, and it can be assumed that a hypothetical distance between the particle and the fluid environment is the hypothetical distance of the extra layer that surrounds the particle. This distance is called the hydrodynamic distance and the potential at this distance is known as the zeta potential. In fact, the zeta potential is a parameter for the potential stability of the colloidal system. If all the particles in the suspension are negatively or positively charged, the particles tend to repel each other and show no tendency to coalesce. www.nature.com/scientificreports/ The tendency of particles to repel each other is directly related to the zeta potential. In general, the limit of the suspension's stability and instability can be determined in terms of zeta potential. Esmaeili and Khodaei 31 reported the negative ZPS of PU. They showed PU can be used as a conductive solution in the double-needled electrospinning method. The negative charge of the PU surface led to a better conduction of ions in the electrospinning device. It also caused better blood compatibility with PU than other polymers. They showed that the negative and positive numbers obtained can be effective in clinical tests 3 . So, we decided to do this test on our sample that contained the three polymers (CS/PLA/PU). The results show that the zeta potential is -1.29 mV, the zeta deviation is 0 mV and the ion conductivity is 13.3 ms/cm. Figure 5 show in vitro release profiles of EOP (A. sativum and Z. officinale) from nanoparticle loaded were investigated in 100 ml of phosphate buffer solution with pH = 7.4 in 48 h. The amount release by UV-Vis spectrophotometry at λ = 275 nm in different ratio scaffolds/EOP (1, 2, 3, and 4) with percent of loading EOP (52.1, 36.2, 28.3, and 24.4%). The higher the ratio of CS/PLA/PU to EOP, the lower the amount of EOP loaded. Therefore, the best formulation is related to the case in which the ratio of CS/PLA/PU to EOP is 1: 1 and has the highest drug load. The release of oils from nanoparticles is done by various mechanisms such as decomposition, desorption and diffusion 32 .

In vitro release of EOP (A. sativum and Z. officinale) loaded.
The mechanism of oil release from the matrix is done by releasing the oil out of the nanoparticles into the environment 33 .The pattern of EOP from nanoparticle release shows a very slow release. For the first initial until final process shows that 75% of the EOP loaded on iron magnetic NPs containing CS/PLA/PU scaffolds was released. The release of EOP at this stage is due to the binding of EOP molecules attached to the surface of the nanoparticle and near the surface of the nanoparticles 34 . It can be attributed to the diffusion of EOP and dispersion in nanoparticles due to penetration into the buffer solution, which causes the bonded CS/PLA/PU with EOP 35,36 . The release of more EOP in the neutral medium may be attributed to the dissolution of the CS/PLA/ PU due to the ionic repulsion of the amino groups present in the CS, PLA, and PU and the binding to other groups 37,38 . In previous research, increase in release to pH conditions related. The release of curcumin from nanoparticles was related to an acidic environment with pH = 5 38 .
Cell viability. In order to evaluate the survival of human fibroblast cells under the influence of [(Fe 3 O 4 @A. sativum and Z. officinale/CS/PLA/PU-Mo-Ge) PEGylated], MTT test was undertaken. Therefore, human fibroblast cells were cultured in appropriate numbers for this test in 24 h after 3D print scaffolds formation. The cells were then treated with a range of drug concentrations in triplicate.
After reading the final absorption by ELISA reader, it was considered to be 100% and other samples were weighed against it. Esmaeili and Hormozi 39 synthesized magnetic NPs of albumin with organic compounds for absorbing and releasing doxorubicin hydrochloride and then investigated the toxicity of NPs by MTT test. Esmaeili and Khalili 3 prepared a scaffold made of CS//PVA/PU with double-needle electrospinning. It contained anticoagulant drugs and we then examined its toxicity using an MTT test. In this study, MTT assay was performed for cancer cells as well as normal cells within 24 h after treatment with the test groups. Table 1 shows

Conclusion
In this study, the CS/PLA/PU were formed into three-3D print scaffolds. Fe 3 O 4 @EOP were attached to these scaffolds and finally their assembly was modified with Fe 3 O 4 @EOP/CS/PLA/PU-Mo-Ge. The finally was PEGylated [(Fe 3 O 4 @EOP/CS/PLA/PU-Mo-Ge) PEGylated]. The best scaffolding was obtained from the concentration of 6% of each polymer in formic acid. In this method, eight vegetable oils were used, all of which are effective in regulating blood pressure. The results showed that A. sativum and Z. officinale oils show similar behaviors. So, we focused most of our work on A. sativum, which showed the best response. EOP used as blood pressure regulators and engineering of cardiovascular tissue. A. sativum and Z. officinale oils show absorption at 275 nm. Therefore, using the UV-Vis spectra, we found that the EOP load on the magnetic iron nanoparticles attached to the 3D scaffold is 75%. Meanwhile, the pH of the working environment was kept at 7.4. Examination of the FT-IR and XRD spectra showed that the 3D print scaffolds of EOP had not chemical interaction. With MTT tests, the absence of cytotoxicity in this system was investigated. The experiment was repeated 3 times in 5 different concentrations with a ratio of about 0.9 for the sample and the control group. It is not clear difference between the sample and the control group, the non-toxicity of the sample was proven. This work is medically biocompatible with blood in the presence of [(Fe 3 O 4 @EOP/CS/PLA/PU-Mo-Ge) PEGylated] due to the structural similarity of CS/PLA/PUwith the layers of blood vessels. It was considered an effective option for cardiovascular systems. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.